Purification And Characterization Of β-1, 3-glucanases From Highland Barley

β-1,3-glucanase[E.C.3.2.1.39]is present in many high plants, encoded by a small gene family. It plays important roles in diverse physiological and developmental processes in higher plant including cell division, microsporogenesis, pollen germination , tube growth, fertilization, embryogenesis and responses to mounding, cold, ozone and UVB, especially it includes in the response of plant microbial pathogens, as well as in the process of germination. Highland barley (Hardeum vulgare var nudum Hook.f) is distinctively grown in Tibetan plateau and QingHai province, China. Although preliminary studies have reported that there were high percents of β-1,3-glucanase in highland barley, the research of purification and function of β-1,3-glucanase from highland barley are absence.In this paper, a major isoform of β-1,3-glucanase from highland barley seedlings was purified following ammonium sulfate precipitation, DEAE-A 25 and CM-cellose ion-exchange chromatography and gel filtration techniques in FPLC , then directly activity stained in polyacrylamide electrophoresis Gels. Identify β-1,3-glucanase speedily, avoiding trivial details of making antibody. The enzyme especially hydraolyzed β-1,3-glucan Laminarin. The molecular weight of the enzyme was estimated about 31 kDa by SDS-PAGE, isoelectric focusing indicated that the β-1,3-glucanase is a basic protein with pI 8.1. Optimum temperature and pH of the enzyme are 35℃ and 5.0. CD spectra techniques was employed to study the secondary and tertiary structure of 8-1,3-glucanase. By analysis the far UV circular dichroism spectra, the secondary structure was estimated as a-helix 14.8%,6-pleatedsheet and p- turn 22.93%, random coil 62.2%. Enzyme in solution under different metal ions was studied by activity assay and fluorescence spectra ,the result show that the highland barley was activated by different metal ions and the divalent ions were effect than sodium at equal ionic strengths. When treated with Na+, no obvious changes of emission intensity and emission maximum ,but treated with Mg2+, Ca2+ and Ba2+,emission intensity were greatly decreased and emission maximum was red shifted specially Ca2+ and Ba2+.These results implied that the binding metal ions changed the microenviroment of aromatic residues in 13-1,3-glucanase. In heat stabilization experiment, this kind of beta-1,3-glucanase was identified as a heat-susptive protein. With increasing of temperature ,it activity descended and obvious change in emission intensity and emission maxium with analysis of. These results implied that irregular change in fluorescence spectra maybe related to its the secondary structure that was high percents of random coil,...